Project Summary/ Abstract Many dsDNA and dsRNA viruses package their genomes into preformed protein capsids using a powerful molecular machine known as a viral packaging motor. One viral motor family, the terminase motor, is associated with both bacteriophage and human pathogens. Terminase motors have three components: the Portal that connects the rest of the motor to the capsid, the Large Terminase (TerL) which has the enzymatic activity of the motor, and the Small Terminase (TerS), which recognizes the viral genome. Although viral genome packaging has been studied for over 30 years, several questions regarding viral motor ATPase mechanism and regulation remain unanswered. Filling these gaps is critical for improving the small-molecule inhibitors that target the motor for treating human pathogens, such as herpesviruses. Additionally, answering these questions will improve the design of the viral motor-powered nanodevices currently being developed as nucleic acid-based therapeutic delivery systems. These gaps have not been properly addressed due of the limitations of commonly used mesophilic model systems. To avoid the shortcomings of mesophilic systems, this project employs an improved novel thermophilic bacteriophage P74-26 model system. This proposal aims to elucidate the mechanism and regulation of TerL ATPase activity. Addressing these gaps in current knowledge will provide insight as to how viral motors translocate DNA during viral genome packaging.